Phosphoinositide 3-kinase (PI3K, also known as phosphatidylinositol-3-kinase and PI3-kinase) plays a critical role in many cellular functions, including survival, proliferation, metabolism, and motility. Cantley L C, Science 296:1655-7 (2002). Dysregulation of the PI3K pathway has been implicated in many human diseases. Hyper-activation of this pathway is known to play an important role in tumorigenesis, whereas deficiencies contribute to the development of type II diabetes. Therefore, this pathway offers promising targets for the development of drugs to combat these diseases.
Class I PI3Ks (α, β, and γ) are recruited to the plasma membrane in response to growth factor and hormone stimulation to mediate the phosphorylation of lipid phosphatidylinositol-4,5-bisphosphate (PIP2), generating phosphatidylinositol-3,4,5-trisphosphate (PIP3), which orchestrates multiple downstream intracellular signaling events. PIP3 signaling is terminated by the phosphatase PTEN, which dephosphorylates PIP3. Genetic alterations targeting PTEN are among the most frequent mutations in human cancers, indicating a critical role of uncontrolled signaling through PIP3 in tumorigenesis and metastasis. Maehama T et al., Trends Cell Biol 9:125-128 (1999). This conclusion is reinforced by transgenic studies establishing that loss of PTEN leads to tumorigenesis. Vivanco I et al., Nat Rev Cancer 2:489-501 (2002).
PIP3 controls a complex cellular signaling network regulating cell growth, proliferation, and survival. PIP3 target proteins are located in the cytosol of unstimulated cells and are recruited to the membrane through pleckstrin-homology (PH) domain-mediated binding to newly formed PIP3. Membrane translocation and activation of the PIP3 target proteins initiate a variety of local responses, including assembly of signaling complexes and priming of protein kinase cascades. PIP3 regulates an array of PH domain-containing proteins, such as serine-threonine kinases Akt and PDK1, GRP1, GDP/GTP exchange factors of Rac and ADP ribosylating factor 6 (ARF6), and protein tyrosine kinases of the Bruton's tyrosine kinase (Btk) and Tec families. Park W S et al., Mol Cell 30:381-392 (2008); Varnai P et al., J Cell Sci 118:4879-4888 (2005); Lietzke S E et al., Mol Cell 6:385-394 (2000). This diversity in PIP3 signaling makes it one of the most important second messengers downstream from growth factor and oncogene signals.
Among the PIP3-controlled signaling proteins, the serine-threonine Akt/PKB (Akt) protein kinase family is of particular interest, because it has been found to play a central role in wide range of fundamental cellular functions including cell survival, growth, and energy metabolism. Datta S R et al., Genes Dev 13:2905-27 (1999); Scheid M P et al., Nat Rev Mol Cell Biol 2:760-8 (2001). The mechanism by which Akt protects cells from death is likely to be multifactorial, involving direct phosphorylation of multiple components of the cell-death machinery such as FOXO transcription factors, BAD, glycogen synthase kinase-3 (GSK-3), and caspase-9. Akt also enhances protein synthesis and cell growth by activating mTOR, which, in concert with another PIP3-binding kinase, PDK1, stimulates p70 ribosomal protein S6 kinase (p70S6K) and inhibits translational repressor eukaryotic initiation factor 4E-binding protein 1 (4EBP1). Cardone M H et al. Science 282:1318-21 (1998); Cross D A et al. Nature 378:785-9 (1995); Datta S R et al. Cell 91:231-241 (1997); McManus E J et al. Nat Cell Biol 4:E214-216 (2002); Tee A R et al. Proc Natl Acad Sci USA 99:13571-6 (2002).
Activation of the Rac family or ARF6 by local gradients of PIP3 plays a major role in remodeling the actin cytoskeleton for directional motility in response to chemotactic agents and growth stimulation. These mechanisms play an important role in enhanced motility of cancer cells and cancer metastasis. Etienne-Manneville S et al. Nature 420:629-35 (2002); Hornstein I et al. Cell Signal 16:1-11 (2004); Venkateswarlu K et al. Biochem J 345 Pt 3:719-24 (2004).
Although lipid-protein interactions mediate PI3K signaling and are frequently deregulated in cancer, most therapeutic strategies targeting the PI3K pathway have focused on inhibitors for downstream targets, including PDK1 and Akt. Peifer C et al., Chem Med Chem 3:1810-1838 (2008); Yang L. et al., Cancer Res 64:4394-4399. Phospholipid-protein interactions have not been as actively targeted, even though lipid molecules are among the most important classes of second messengers.
We recently discovered selective non-phosphoinositide small molecule PIP3 inhibitors, termed PITenins (PITs). US Pat. App. 2012/0016033 A1; US Pat. App. 2013/0036033; Miao B et al., Oncogene 31:4317-4332 (2012). PIT-1, selected in a screen of about 50,000 small molecules by using a PIP3/Akt PH domain binding assay, and its derivative DM-PIT-1 have been extensively characterized. PIT-1 and DM-PIT-1 effectively inhibit cancer cell survival and induces cell apoptosis by specifically inhibiting PIP3-dependent PI3K-PDK1-Akt signaling, resulting in significant antitumor activity in vivo.